The fight against the intolerable burden of malaria is restricted to the use of insecticides that kill the mosquito vector and drugs that kill the parasite in humans. Considering that recent estimates place the number humans infected with malaria at over 500 million (nearly 1 in 12 humans), the introduction of new means to counter the disease is urgently needed. We propose to develop a novel strategy to prevent the spread of malaria parasites by anopheline mosquitoes, based on genetic modification of bacteria that inhabit the gut of these insects. We will evaluate in detail the ability of two candidate bacterial species (Pantoea agglomerans and Asaia sp.) to colonize larval and adult mosquitoes and their ability to be transmitted vertically from one generation to the next. We will develop robust methods to secrete antimalarial effector proteins from each bacterial species to ensure that the effector proteins will reach the intended targets on the parasite or on the midgut epithelium. Different bacterial strains that secrete effector proteins will be evaluated for their efficacy to interfere with parasite development in the mosquito. Using the data obtained from engineered bacterial strains producing single effectors, we will create an optimal combination of strains that combine multiple effectors for maximum efficacy. Given that this funding mechanism is restricted in its time frame, no field trials are proposed although discussions with the relevant regulatory agency (US-EPA) are already underway. This research is expected to lead to the development of a novel weapon that can be used in combination with traditional control strategies (drugs, insecticides, vaccines) to combat malaria. PUBLIC HEALTH RELEVANCE: Malaria is one of the deadliest infectious diseases and kills an estimated 2 million persons every year. The mosquito is the obligatory vector for transmission. This project will devise new ways to interfere with the mosquito capacity to transmit the parasite, by genetically modifying bacteria that live in the mosquito midgut.